Proteolytic enzymes, their production and use

ABSTRACT

The invention relates to enzyme preparations containing novel proteolytic enzymes produced by cultivation of novel species of the genus Bacillus and showing useful activity at high alkalinities. The invention also relates to production of the novel proteolytic enzymes by cultivation of the novel species of the genus Bacillus in a nutrient medium having a pH-value within the range of 7 to 12. Finally the invention relates to a process of isolating the novel species referred to by effecting the isolation on nutrient media having a pH-value within the range of 9 to 11. The novel enzymes are particularly useful in detergent and dehairing compositions.

United States Patent 1 Aunstrup et a l.

[ 51 Mar. 27, 1973 PROTEOLYTIC ENZYMES, THEIR PRODUCTION AND USE inventors: Knu d Aunstrup, Farum; Otto Andresen, Copenhagen; Helle Outtrup,

Vaerlose, all of Denmark [73] Novo Tera'peutisk Laboratorium A/S, Copenhagen, Denmark Filed: Sept. 23', 1968 Appl. No.: 761,546

Assignee:

[30] Foreign Application Priority Data Oct. 3, 1967 Great Britain ..45,046/67 US. Cl. ..1 95/62, 195/65, 195/29 Int. Cl. ..Cl2d 13/10 Field of Search ..195/62, 63, 65, 66, 68, 63 P,,

195/118;210/l1;252/DIG.1i

[56] References Cited OTHER PUBLICATIONS MAXATASE, published by Royal Netherlands Fermentation Industries Ltd., Delft-Holland, April I967. Alcalase, published by Novo lndustri, Copenhagen, Denmark, 1969 pp. 2-5.

Primqry Examiner-Lionel M. Shapiro Att0rney-Synnestvedt & Lechner [57] ABSTRACT process of isolating the novel species referred to by ef-.

fecting the isolation on nutrient media having a pH value within the range of 9 to 11. The novel enzymes are particularly useful in detergent and dehairing compositions.

4 Claims, 3 Drawing Figures PATH'HIZDHAR27 1975 f/oAktivity TYPE 1 (c 311 1 pH 7 a 9' 10 11 12 1. Ak1iv11 TYPE '2 (c 335) l I l 7 a 9 10 11 12 f/oAktivity TYPE 3 c324) 7' a H 7 a 9 10 11 12 p PROTEOLYTIC ENZYMES, THEIR rnonuc'rrou nn USE This invention deals with preparations and compositions comprising new enzymes showing a pronounced and useful proteolytic activity at high alkalinities.

Within various fields there is demand for proteolytic enzymes and in some of these fields it is important that the enzymes display optimal proteolytic activities at high pH-values up to to 12 and even higher, and that the enzymes are in possession of other useful properties, such as stability at elevated temperature and/or stability in the presence of non-enzymatic substances being a constituent part of the enzyme-containing preparations or compositions.

it is known that proteolytic enzymes can be produced by cultivation of certain bacteria under aerobic conditions, but the proteolytic enzymes produced by the known cultivations show optimal proteolytic activity against hemoglobin at a pH-value which in the most favorable cases did not reach more than 9.

The present invention is based on the observation that there exists in nature a great number of hitherto unknown bacteria forming during their metabolism proteolytic enzymes which display optimal proteolytic activity against hemoglobin at high pH-values up to 10 to 12, and which have other properties making them excellently suited for use within different industrial fields.

From samples of soil, animal manure and a number of other sources in nature the inventors have isolated about one hundred strains of bacteria, carried out taxonomic investigations and found that all of the hitherto unknown bacteria belong to the genus Bacillus, but that none of them belonged to any species known to the inventors, and that, to the best of the inventors knowledge, they did not belong to the same species. Furthermore, within the same species there were in most cases different strains and several varieties.

For the purpose of isolating the hitherto unknown bacteria referred to above, use has been made of a novel technique which is characterized by the fact that the isolation is effected on nutrient media having a pH- value within the range of 9 to 11, and intended for detection of production of proteolytic enzymes.

In other words, the samples of soil, animal manure or other sources from nature have been spread on nutrient media having the high pH-value referred to and the bacteria able to grow under such alkaline conditions are then isolated and subjected to further investigations as to species and enzyme production.

In most cases, use has, according to the invention, also been made of a number of different enrichment methods.

Enrichment methods are known in the art. Reference can be made to Hayaishi, Methods in Enzymology, Vol. 1, 126-131. One principle is to let a sample from nature grow on a nutrient medium having a specific and selected composition favoring the-growth of a microorganism giving metabolic products having the properties aimed at. Another principle is to store the sample from nature together with a compound, such as an inorganic salt, favoring the development of the desired microorganism, cfr. M.A. El-Nakeeb and H.A-.Lecheva1ier, Appl. Microbiol., Vol. 11, 75 (1963), and thereafter to spread the sample on a suitable nutrient medium adjusted on a pH-value within the range of 8 to 12.

Some of the hitherto unknown members of the genus Bacillus which have been isolated and tested-taxonomicolumn the enrichment method used.

TABLE I Ref. Number N o. N 0113 Source of isolation Enrichment method 0 300. 10144 Soil from cemetery Starch-casein medium in Copenhagen. (pH raised stepwise from 10 to 12).

C 301.- 10145 ....-do

C 302. 10146 Soil from Copenhagener Spreading of soil samples river bank. on agar with sesquicar- C 303. 10147 Heap of soil and leaves bonatc (pH=9.6-0.8).

from cemetery in Testing 01' zones of Copenhagen. hydrolysis on neutral C 304. 10148 Wood sand from agar with skim milk.

Blokhus, Jutland.

C 311. 10281 Wood soil from Asche- Perborate agar.

berg, Holstein.

C 323. 10282 Field soil from Danish Do.

town.

C 324. 10283 Lake bank soil from Do.

Ascheberg, Holstein.

C 325. 10284 Infection on plate with Alkaline skim milk agar perborate holes. plates with holes filled C 326. 10285 ....do with sodium pt-rhorate.

C 334. 10286 Bank river soil from Perborate agar.

Danish town.

C 335. 10287 Garden soil from Do.

Danish t wn.

C 336. 10288 Horse and elephant Do. manure.

C 337. 10289 Clay from grass field Ierhorate storing.

from Ascheherg,

Holstein.

C 338. 10290 Soil from cemetery in Do.

Copenhagen.

C 330. 10201 Bank river soil from lerborate agar.

Danish town.

C 341.. 10293 Field soil from Danish Do.

C 342. 10204 Garden soil from Soda storing.

Danish village.

C 343. 10205 Garden soil from lerborate agar.

Danish town.

C 346. 10296 Chicken yard soil from Multiple alkaline starch Danish town. enriclnncnt.

C 347. 10207 Deer manure from deer Do.

park near Copenhagen.

C 348. 10208 Chicken run soil C 340. 10200 Deer manure from deer I) park near Copenhagen.

C 350. 10300 Waterfront Copen- Alkaline starch-casein hagencr lake. medium with tripolyphosphate.

C 351. 10301 Chicken inanure 'Iherrnophile sesquiearhonate enrichment C 352. 10302 Ostrich manure lroin Sodium sesquicarhonato Zoo. enrichment (pH Lil-0.6).

C 353.. 10303 Manure from elephant o.

C 354. 10304 Chicken yard soil Enrichment on basic glucose nitrate at 40 C.

C 355.- 10305 Do.

C 356. 10306 Garden bark l'lnriehnn-nt on basic glucose nitrate at 50 C 357. 1010? Chicken yard soiL. Do.

(I 360. soil from lerlmrnl agar.

Danish town.

C 364. 10310 Lavatory cistern 'lherniophile sesquicar- Scraping. lmnate enrichment (50 p11; spa-0.7).

C 365. 10311 Liquid from tannery Bran-soda enrichnn-nt.

liming hath.

(. 366. 10312 Baby faeces Starch enrichnn-nt pll 11) with inorganic nitrogen.

C 367. 10313 Manure from l-lephanL. Therrnophile sl-squieurlmnatc enrichment (50 (.1, pH; 8.8-0.7).

C 360., 10314 Ostrich manure from lroteose peptone (Shake zoo. flasks) p11 1.7.

C 370. 10315 Scraping from tanneiy Alkaline niannitol-KNOa liming bath enrichment. containers.

C 371. 10316 Manure from elephanL. lroteose peptonr- (shake llasks) pH 0.7.

TABLE 1-Continiied Ref. Number No. NCIB Source of isolation Enrichment method C 372. 10317 Clay from grass field Starch casein detergent from Aschcberg, enrichment. Holstein. C 373. 10318 Garden soil from Pcrborato agar.

Danish town. C 374. 10319 Clay from grass field Ethylene diaminc tetrai'roni Aschcberg. acetic acid sodium salt Holstein. starch casein enrichment. C 375. 10320 Ostrich manure from Sodium scsquicarbonatc zoo. enrichment (pH; 9.2-

0.6). C 376. 10321 Manure from elephant Do. (J 377. 10322 Water from hippo- Tlierinophilc casein starch potamus basin. enrichment with IfIaOH. C 378. 10323 Scraping from tanner MannitoI-KNO: t t

liming bath mcnt. containers. C 410. 10324 Tiger manure" Tll(l'lll0[)llllt StSqlllCtlY- C 411. 10325 PlgtOil1Ii21lllll'0 hon-ate enrichment (50 (h, pll; 8.8-9.7). C 412. 10326 Chicken yard soil from lutato Hour and sodium Danish town. scsquicarlmnatc storing. C 413. 10327 Clay from grass field Starch enrichment (pH 11) from Asclu'hoi'g, Holstein.

the methods described by Smith, Gordon & Clark in Aerobic Spore-forming Bacteria," U.S. Department of Agr., Monograph No. l6 (1952). These methods are up till now considered the most suitable ones, but they had to be modified in view of the fact that all nutrient media had to be adjusted on a much higher pH-value than that indicated by Smith, Gordon & Clark because all the Bacillus species listed in Table I grow at elevated pH-values.

The bacteria can be divided rather accurately into morphological groups. These groups differ from each other to such an extent that they actually represent separate species.

Within the morphological groups variations in the biochemical reactions are found. On the basis of these variations the groups have subdivided into varieties which are represented by one or more strains.

SPECIES NO. I (belonging to morphological group I according to Bergey) Morphology Vegetative rods: 0.5 0.7 p. X 1.5 4 u Spores: 0.5 0.8 X 0.8 1 u central to subterminal, oval to cylindrical, thinwalled.

Sporangia: Very little, if any, swelling of spores.

Variety a: C 300,C 301, C 360, C 372, C 374 Grampositive.

Growth or nutrient agar pH 7.3 as good as or better than on nutrient agar pH 9.7.

Maximum temperature for growth 505 5C.

Scant growth, if any, on glucoseor mannitolagar pH 9.7 with nitrate as sole source of nitrogen.

Hydrolysis of starch: Positive, narrow zone of hydrolysis after 7 days.

Variety b: C 302, C 334.

Grampositive.

Growth on nutrient agar pH 7.3 rather slow during the first two days, thereafter nearly comparable to growth on nutrient agar pH 9.7.

Maximum temperature for growth 4050C.

Scant growth, if any, on glucoseor mannitolagar pH 9.7 with nitrate as sole source of nitrogen.

with inorganic nitrogen.

Hydrolysis of starch: Positive, wide zone of hydrolysis.

Variety c: C 323, C 339,C 352,.C 369.

Grampositive or Gram variable. Moderate to scant growth on nutrient agar pH 7.3. Maximum temperature for growth 3750C. Scant growth, if any, on glucoseor mannitolagar with nitrate as sole source of nitrogen.

Hydrolysis of starch: Positive, wide zone of hydroly- SIS. Variety d: C304,C 31 l,C 336.

Grampositive rods. Moderate to scant growth on nutrient agar at pH 7.3. Maximum temperature for growth: 37C. Scant growth on glucoseor mannitolagar at pH 9.7 with nitrate as sole source of nitrogen.

Hydrolysis of starch: negative.

SPECIES NO. [I (belonging to morphological group I according to Bergey) Morphology:

Vegetative rods: 0.3 0.4 p. X l .5 2.5 u Spores: 0.3 0.5 p. X 0.8 l u central to paracentral, oval to cylindrical, thinwalled.

Sporangia: Very little, or no, swelling by spores.

Grampositive Almost no growth on nutrient agar at pH 7.3

Maximum temperature for growth: 37C I No or scant growth on glucoseor mannitolagar with nitrate as sole source of nitrogen.

Hydrolysis of starch: Negative.

SPECIES NO. III

Morphology:

Vegetative rods: 0.6 0.7 p. X 1.5 3.5 p.

ends rounded.

Spores: 0.7 0.9 a X 1.0 1.2 u ellipsoidal, paracentral thickwalled.

Sporangia: Some are definitely swollen, others are not. The rods in sporulated cultures swell and grow very thick but keep their original shape. Sporulating short rods may have a globular form at this stage. Thus, there is no local swelling at the site where the spore is placed, although some sporangia are spindle-shaped.

Variety a: C 326, C 342.

Grampositive.

No or only scant growth on nutrient agar at pH 7.3.

Maximum temperature for growth: 50C.

No or only scant growth on glucoseor mannitolagar at pH 9.7 with nitrate as sole source of nitrogen.

Hydrolysis of starch: positive, wide zone of hydrolyto subterminal,

Variety b: C 347, C 350.

Grampositive. Moderate growth on nutrient agar at pH 7.3. Maximum temperature for growth: 50C. Scant growth on glucoseor mannitolagar at pH 9.7 with nitrate as sole source of nitrogen.

, with nitrate as sole source of nitrogen.

Hydrolysis ofstarch: positive, wide zone of hydrolysis. Variety c. C 337, C 340.

nitolager at pH 9.7 with nitrate as sole source of Moderate growth on glucoseor mannitolagar at pH 9.7 with nitrate as sole source of nitrogen.

Hydrolysis of starch: positive, wide zone of hydroly-f SIS.

SPECIES NO. IV (belonging to morphological group ll according to Bergey).

Morphology Vegetative rods: 0.4 0.5 a X 2-3 ,L, often in long chains.

Spores: 0.6 0.8 p. X 0.7 0.9 u,

oval, subterminal, thickwalled, easily stained. Sporangia: Definitely swollen, clavate. Variety a: C 303, C 354, C 357, C 366, C 367, C 371, C 375, C 378.

Gramnegative.

Moderate to good growth on nutrient agar at pH 7.3. Maximum temperature for growth: 57C.

Moderate to good growth on glucoseor mannitolagar at pH 9.7 with nitrate as sole source of nitrogen.

Hydrolysis of starch: positive, moderate zone of hydrolysis. Variety b: C 351,C 356,C 364,C 376,C 377,C4l1

Gramvariable. Otherwise like variety a. Variety c: C 358, C 410.

Grampositive. Otherwise like variety a.

SPECIES NO. V (belonging to morphological group I1 according to Bergey) Morphology Vegetative rods: 0.3 0.4 p. X 2-4 p, straight, some slightly bent Spores: 0.6 0.7 p. X 0.9 1.2 it,

oval to ellipsoidal, paracentral to terminal,

thickwalled, easily stained, remnants of sporangia often adhering.

Sporangia: Definitely swollen, clavate to racketshaped. Grampositive.

Moderate to good growth on nutrient agar at pH 7.3.

Maximum temperature for growth: 57C.

Good growth on glucoseor mannitolagar at pH 9.7 with nitrate as sole source of nitrogen.

Hydrolysis of starch: positive, moderate to wide zone of hydrolysis.

SPECIES NO. Vl (belonging to morphological group II according to Bergey) Morphology Vegetative rods: 0.25 0.35 ,t x 2.5 5 ,1.

Slightly bent, ends pointed Spores:O.8-1 ;;.X 1.1 1.3 [.L.

Oval, to ellipsoidal, subterminal to terminal.

Sporangia: Definitely swollen, clavate to drumstickshaped. Variety a: C 373 Gramnegative.

No or only scant growth on nutrient agar at pH 7.3.

Maximum temperature for growth: C.

No or only very scant growth on glucoseor mannitolagar at pH 9.7 with nitrate as sole source of nitrogen.

Hydrolysis of starch: Positive, wide zone of hydrolysis. Variety b:

Grampositive.

' Moderate growth on nutrient agar at pH 7.3.

Maximum temperature for growth: 50C.

No or only very scant growth on glucoseor mannitolagar at pH 9.7 with nitrate as sole source of nitrogen. I

Hydrolysis of starch: Positive, wide zone of hydrolysis.

SPECIES NO. Vll

This strain was tentatively classified as a separate species.

It is asporogenous or oligosporogenous and therefore cannot be placed in any morphological group.

It seems mostly related to the'alcaline bacteria which belong to the morphological group ll of the genus Bacillus (according to Bergey).

Vegetative rods: 0.3 0.5 p. X 2 5 t,

often in long chains, filamentous, ends slightly pointed and rounded. Gramnegative.

' nt arat H7.3. m Moderate to good growth on nutrie ag p d C 338, C 343 C 346, Maximum temperature for growth: 57 C. C 348' C 349 Good growth on glucoseor mannitolagar pH 9.7 e c 3z4,c 355 wi th nitrate as sole source of nitrogen. g g C 354 C 357 Hydrolysis of starch: Positive, moderate zone of C c c hydrolysis. C 375.C 378 The following characters are common to a number of W b C 351' C 356 C 364 strains belonging to the different varieties in the species C 375' c C 411 I-IV: c C358,C4l0

v 10 C 365, c 412 a C 373 Hydrolysis of gelatme: Positive v Hydrolysis of casein: Positive. v" b g C Glucose-nutrient agar slants Growth same as on nutrient agar, or heavier. All the strains referred to have also been grown on Soybean agar slants Growth more abundant and nutrient agar and soybean agar and some of them on softer than on nutrient agar. glucose nitrate agar and mannitol nitrate agar, for the Tyrosine agar slants Growth same as on nutrient purpose of observing the form, appearance and color of agar. the colonies, but as the relevance of these observations Nutrient broth Medium turbidity with abundant may be disputed, the results of the inventors observasediment. No pellicle or pellicle thin and friable. tions are not reported here. NaCl broth Good growth in 5 percent concentra- However, the following Table III reviews the propertion of NaCl; growth at 7 percent. ties the variations of which form the basis for the divi- Production of acetylmethylcarbinol Negative. sion of varieties within the different morphological Reduction of nitrate to nitrite Positive. groups:

TABLE III Width of Growth on zone of Growth on Maximum nitrate (N03 Hydrohydrolysis Maximum Gram nutrient growth as sole N- lysis of on starch growth velo- Species Var stain agar pH 7.3 temp. source starch agar city at pH I a 50-55 Scant l) -50 Scant e 37-40 Seant d 37 Scant II Scant 37 Seant III a Scant 50 Scant b 50 Scam c var. 37,50 Seant d var Scant -50 Scent e Scant 45-50 I IV a 5 8.09.0 1) var. s7 8.0-9.0 C 57 VI a Seant 50 Scant 8.5-9. 2 b 50 Scam. VII 57 8.0-8.5

NoTE.--Seant =po0r; Stain=colour.

Anaerobic growth in glucose broth Scant, if any, growth; pH 7.8 or higher at 14 days. (pH of the medium adjusted to 9 before inoculation).

On the basis of the inventors taxonomic investigations the members of the genus Bacillus listed in Table I should be classified as it appears from Table II below.

TABLE 1 amount of proteolytic enzyme digesting hemoglobin at 60 a pH-value of 10.1 and a temperature of 25C during a p g y figf 301 C 360 reaction time of 10 minutes with such an initial velocity C C 374 that per minute there IS formed such an amount of split 1 b C 302,C 334 products which cannot be precipitated with c gig; C C trichloracetic acid that these split products give the d c 3o4,c31|,c 336 same color with phenol reagent as does one mila g :2; liequivalent of tyrosine.

b c 3471C 35 The process for producing the proteolytic enzymes c C 337,C 340 belongs to the known art in which the enzymes are produced by aerobic cultivation of bacteria in a nutrient medium containing assimilable carbon and nitrogen sources, and the characteristic feature of the inventive process consists in maintaining a pHvalue of the nutrient medium within the range of 7-12 during the cultivation, using species of the genus Bacillus able to grow and' produce the proteolytic enzymes within the pH-range referred to, and recovering from the medium the proteolytic enzymes formed during the cultivation.

Experiments seem to show that it is convenient to carry out the cultivation at a pI-l-value of the culture mediumwithin the range of 7.5 to 10.5.

The nutrient medium is composed in agreement with the principles of the known art. Suitable assimilable carbon sources are carbohydrates, such as saccharose, glucose, starch, flour from cereal grains, malt, rice, sorghum etc. The carbohydrate concentration may vary within rather great limits, e.g., up to 25 percent and down to l percent, but usually 8 10 percent would be suitable, the percentage being calculated as dextrose. It has been found that the presence in the nutrient medium of carbohydrates will give rise to the formation of acidic components, resulting in a decrease of the pH-value during the cultivation. As it is essential to maintain a pl-l-value of the nutrient medium within' the range of 7 to 12 during the cultivation, measurements should be taken that the pH-value does not fall below 7 or any essential period during the cultivation. In order to keep the pH-value within the required range, a limited amount of carbohydrates may be used together with a buffer substance which is able to maintain the required pH-value. It has been found that carbonates, and particularly sesquicarbonates, used in a concentration of up to 0.2 M in the medium, is able to create a pH-value of about 10.5 and 9.3, respectively.

Also other buffer systems, such as phosphate buffers, may be used.

It is also possible to initiate the cultivation with a low carbohydrate content and to add small amounts of carbohydrates successively during the cultivation.

A third possibility is to make use of automatic pH- control by addition of various basic-reacting substances used in this art.

The use of carbonates and sesquicarbonates as pH- controlling substances is very useful and it is surprising that it is possible during the cultivation to use these compounds in the concentrations referred to.

The nitrogen source in the nutrient medium may be of inorganic and/or organic nature. Suitable inorganic nitrogen sources are sometimes nitrates and ammonium salts, and among the organic nitrogen sources there are quite a number known for use in fermentation processes and in the cultivation of bacteria. Illustrating examples are soy meal, cotton seed meal, peanut meal, casein, corn steep liquor, yeast extracts, urea, albumine, etc.

Besides, the nutrient medium should naturally contain the usual trace substances.

The temperature at which the cultivation takes place is normally within the same range as in the known cultivation of known species of the genus Bacillus. Usually a temperature between and 55C is convenient. The temperature is preferably to C.

As the cultivation has to be carried outunder aerobic conditions, it is, when using fermentation tanks, necessary to make use of artificial aeration. The amount of air is similar to that used in the known cultivation processes.

In general, maximum yields of the proteolytic enzymes will be obtained after a cultivation time of l to 5 days.

Although most of the experiments in connection with the production of proteolytic enzymes from the species and strains compiled in Table II, have been carried out in shake flasks or in tanks in pilot plants, use has also been made of surface growth. in such case the nutrient medium consisted of 10 g of wheat bran, 2 g Na PO 12l-l,0 and about 10 ml water. Prior to inoculation the pH-value was adjusted to about 10 with 2 ml 1 N NaOH. On this medium strains C 300 and C 303 were cultivated by surface growth, and for both strains the yield of proteolytic enzymes was about 20 Anson units per kg of wheat bran at pH 10.

For cultivation of the species and strains compiled in Table II the following two media were used:

1. Medium BPFA with the following composition:

Potato flour 50 g per liter of tap water Saccharose 50 g Barley flour 50 g Soy meal 20 g Sodium caseinate l0 g Na,HPO l2H,O 9 g Pluronic 0.1 g

2. Medium BSX with the following composition:

Barley flour g per liter of tap water Soy meal 30 E l I Pluronic 0.1 g

Both these media were adjusted to the desired pH- value by the addition of sesquicarbonate or soda under sterile conditions.

The experiments in shake flasks were carried out in 500 ml shake flasks, each of the flasks containing 100 ml of the nutrient medium BPFA and BSX, respectively, which were sterilized beforehand by autoclaving for TABLE IV Sp. Var BPFA BSX Anson Ansom units pH of units pH of Strain per kg medium per kg medium C 300 19 6.5 19 9.5 C 301 10 6.5 24 9.5 a C 360 20 6.3 30 9.5 C 372 20 6,4 15 9.3 C 374 ll 6.3 l8 9.3 b C 302 36 9.2 16 9.3 l C 334 38 8.6 10 9.5 C 323 22 7.6 9 10.0 C 339 38 9.3 19 9.2 C 352 32 9.1 33 9.5 c C 369 l 15 9.3 16 9.3 C 304 48 8.1 7 6.5 d C 311 67 9.4 ll 97 C 336 2 8.0 l 9.1 ll C 335 38 9.5 3 10.0 C 341 42 9.2 3 9.9

1 1 12 c 303 3 7.2 30 9.7 addition before C 354 17 9.0 11 9.1 inoculation) ad 0.2 M 0.2 M 0.2 M 0.4 M C 357 4 8.2 25 9.4 pH before inocu- C 366 5 8.7 30 9.5 lation 10.0 10.0 10.35 10.1 a C 367 4 8.8 35 9.4 Cultivation IV C 371 2 8.5 40 9.4 temperature C 34 34 34 34 C 375 l 7.6 30 9.6 5 Air mlminutc 0.3 0.3 0.3 0.3 C 378 2 8.5 25 9.3 Cultivation time C 351 2 8.3 30 9.7 in hours 125 104 H3 126 C 356 3 7.7 20 9.7 pH at maximum 9.3 9.3 9.6 9.3 b C 364 3 8.3 40 9.3 Maximum Anson C 376 9.3 20 9.7 units per kg 67 80 77 66 O The proteolytic enzymes can be recovered from the c C 35 2 25 9,6 cultivation broth by subjecting the broth to centrifuga- C 0 3 17 tion, precipitating the enzyme from the liquid thus ob- V gig 3 g3 95 tained by addition of Na SO or ethanol, separating the v" C 370 H5 90 45 93 precipitate from the liquid by filtration with kieselguhr 8 Egg; 2' 3-; i 3': as filtration aid and drying the precipitate to form a owder containin the active roteol tic enz mes. b c347. 44 9.3 7 9.1 P g P Y C 350 44 8.8 13 8.9 These recovery processes belng considered as examc Egg; 3'2 l 3'? ples, only, may be illustrated by reference to Table Vll 111 c 338 72 8:2 7 7:7 below C343 14 9.0 2 8.9 d c 346 26 3.5 5 9.4 TABLE V C 348 17 9.6 7 9.l e Strain: c 339 c 347 c351 C355 60 6 I Starting 250 kg culture 250 kg culture 600 ml culf C 353 96 8 material liquid containliquid containture liquid 3 C 373 '2 1 ing Anson ing 45 Anson containing V. b C 325 1 1 25 units per kg units per kg 32 Anson C 413 0 18 units per liter Total amount The two culture media BPFA and BSX have also o on r units 6250 11250 19.2 beenused for cultivation in tanks under submerged Cemrb 3000 rpm/3O 3000 rpm/ 4000 conditions and artificial aeration, use being made of 30 fu ati n i in 30 min. 550 liter stainless steel tanks. To illustrate such cultiva- F P N so N so 2% l a lon g a 4 g a, 4 m trons in p lot plant reference [8 made to the following Standing one Standing one CHBOH Table V giving information of the strains used, the culhour hour tivation conditions and the results obtained. E kg Klese" Press 35 "on guhr filter (centlgfugation 3 min. TABLE v 4000 pm washin with Strain c 324 c 335 c 339 c 347 c 351 600 rnl c,ll,ou Medlum BPFA BPFA BPFA BPFA BSX move centrifugapH-value before I ig gfig g inoculat on 9.3 10.3 10.5 10.2 10.2 Drying Drying cham Drying charm vacuum, l ber 40C ber 40C P,o, fi it Celslus 34 34 34 34 34 Enzyme 4000 g con- 1300 g con- 9.7 g conf m3 er powder taining 0.4 taining 0.8 taining 1.3 rnil'lute p 0.3 0.25 0.25 0.25 0.3 8:52 93 22 Cultivation time in hours 53 84 97 59 83 Yield 1600 Anson 1020 Anson 12.6 Anson Final pH-value 8.3 9.2 9.1 3.9 9.35 25% 33 22%; Final proteolytic activity exs fi zg ggi The starting materials mentioned in Table Vll have substrate 44 40 29 4s 33 been produced by cultivation in 550 liter stainless steel tanks under artificial aeration. The nutrient media were adjusted on the initial pH-value by a 2 M Na CO solution added under sterile conditions before the inoculation.

Testing of the proteolytic enzymes produced by the strains listed in Table ll has shown that there are material differences between the enzymes regarding a number of their properties.

With regard to the proteolytic activity it seems that the enzymes may be divided into three groups or types T BL Vl when the proteolytic activity is measured at pH 12 and ex ressed in ercenta e of maximum activit viz. figf' 2 3 4 Type 1: 108 to 80 y Barley flour Type 22 I0 50 gagged 100 200 Type 3: 50 to 0 l 30 30 45 60 It is known in the art that calcium ions stabilize the 2 3L221 0m 0403 003 003 activity of most of proteolytic enzymes. The novel on- Na CO (sterile zymes produced by the bacteria listed in Table and diresults of-the enzyme type testing and the calcium ion stabilization effect are compiled in Table VIII, in which plus means that the residual proteolytic activity in the absence of calcium ions is below 80 percent of the corresponding activity of the control in the presence of TABLE VIII Species Var.Strains: Enzyme-type Ca stabiliza tion a C300,C3OI,C360, l -H-l-l-l- C 372, C 374 I b C 302, C 334 l c C 323, C 339, C 352, 2 ?+H- C 369 d C304,C3II,C336 l 4-H II I C 335, C 341 2 -H- a C 326, C 342 3 b- C 347, C 350 3 c C 337, C 340 3 +1- Ill d C 338, C 343, C 346, 3 +7??? C 348, C 349 e C 324, C 355 3 f C 353 3 C 303, C 354, C 357, l C 366, C 367,C 37l,C 375} C 378 IV b c 351, c 356, c 364, 1

C 376, C 377, C 411 c C 358, C 410 l V' C365,C4l2 l 21 C373 1 VI b C 325, C 413 I 7? VII C 370 l The proteolytic activity of the enzymes produced by the strains listed in Table VIII has been tested not only at pH 12, but also at lower pH-values to give a more detailed impression of the proteolytic activity at different pH-values and more information of the activity of the three types of enzymes.

For the purpose of illustration reference is made to the drawings on which FIG. 1 shows the proteolytic activity of the enzyme produced by the strain C 311, said enzyme belonging to type 1,

FIG. 2 in the same manner showsthe activity of the enzyme produced by strain C 335, said enzyme belong-' ing to type 2, and

FIG. 3 in the same manner shows the activity of the enzyme produced by the strain C 324, said enzyme belonging to type 3.

It should be understood that the purpose of the activity curves shown on the drawings is to illustrate in principle the difference in activity of the three types of proteolytic enzymes at varying pH-values and that the activity curve for each type may vary somewhat without missing its characteristic appearance.

The novel enzymes according to the invention have furthermore been subjected to the following tests:

a. Stability against tripolyphosphate (TPP).

The stability of the enzymes in a solution containing tripolyphosphate in an amount of 0.2 percent was determined. The stability has been expressed as per-' centage of residual activity after minutes at C and at pH 10. The enzyme concentration was 0.1 Anson unit per liter and the method of analysis was the Anson method. 1

The results are compiled in the following Table IX.

The corresponding solutions with 0.01 M CaCl showed in all cases a residual activity of to percent. b. Stability against perborate The activity of a solution containing the enzyme and sodium perborate in an amount of 0.1 percent was determined. The stability was expressed as percentage of residual activity after 30 minutes at 50C and pH 10. The enzyme concentration and the method of analysis were as in test a).

The results are compiles in the following Table 1X. c. Stability against surfactants The stability of solutions containing the enzyme and different surfactants was determined while using three typical surfactants in concentrations corresponding to those employed in the washing solution:

1. Soap 0.25 g per liter 2. DBS an alkylarylsulphonate (50%) 2.5 g per liter 3. TAS -tallow-alcoholsulphate (25%) 5.0 g per liter.

The enzyme concentration was 0.1 Anson unit per liter. The test conditions were 30 minutes at 50C at pH 10, and the method of analysis was the nitro-caseinmethod, cfr. E.v. Pechmann, Biochemische Zeitschrift, Bd. 321, 248-260 (1950).

The figures inTable IX are to be understood as follows:

The figure above the stroke shows the percentage of residual activity, when one performs the analysis immediately after the addition of the surfactant, i.e., this figure gives an indication of the initial rate of inactivation.

The figure under the stroke is the difference between this initial residual activity and the percentage of residual activity after 30 minutes.

(1. Temperature optimum In Table IX is indicated the temperature indegrees of Celsius at which there was found maximum activity at pH 10. The method of analysis was the Anson method.

e. Enzyme type It has been found that all the enzyme preparations are inhibited momentaneously and completely by.

Testing of the enzyme preparations produced by strains C 303 and C 347 against sodium sulphite has shown that the enzymes are not sensitive to this reducing agent, which might indicate that S-S-bridges are not essential for the tertiary structure of the enzymes.

TABLE IX Stability Enzyme preparation Activity. Surfactants Temp. in powder Anson units Enzyme Peroptimum form, g. per g. at pH type TPP boratc Soap DBS TAS C. Serino pH stability 2 0. 5 (10) 1 2 89 82/71 56/48 7/6 50 2 0. 7 (10) l 4 47 50/47 35/31 2 50 3 0. 7 (10) 1 1 48 551/56 60/ 3/1 50 000 3. (7. I III 05 85/50 40/30 23/18 (30 2. 5 0. 3 (7. 5) l .3 4 57/50 34/31 3/0 50 I l. 500 0. 5 (T. .5) I J5 13'.) 100/83 00/27 03/73 I it I. 3 (7. 5) I 7H tili 03/67 (37/50 53/45 till I II. 0. ti (7. 5) 1 EH .3 77/33 60/38 00/53 I30 I- 1 7 0. 4 (7. 5) I 0 83 07/04 76/70 75/75 50 450 0. I (7. 5) I 00 80 81/31 27/11 10/8 00 .2, 000 0. I (7. 5) I 04 86 100/0 72/20 02/48 00 26 0. 8 (7. 5) 1 100 06 02/42 67/14 68/61 60 500 2. 2 (7. 5) 1 I14 100 88/2 58/34 71/52 60 3, 000 0. 6 (7. 5) 1 89 01 94/4 33/15 67/30 60 50 1. 0 (7. 5) 1 95 03 07/60 70/44 93/57 60 430 2. 7 (7. 5) 1 6 60 98/05 75/68 87/85 50 17 7. 8 (7. 5) 1 88 78 66/42 25/10 /12 60 213 1. 0 (7. 5) 1 J3 86 100/10 59/27 74/50 60 1, 500 0. 3 (7. 5) 2 60 05/51 40/32 78/71 55 217 1. 4 (7. 5) 2 16 76 80/10 75/57 83/62 45 7. 2 1. 4 (7. 5) 2 81 2 28/28 6/6 0/0 50 573 2. 0 3 0 36 81/78 94/71 67/63 40 From Table IX it will be seen that a number of the enzyme preparations show astonishing stability properties.

In general, the enzyme preparations or compositions according to the invention consist of a solid or liquid mixture of the proteolytic enzymes produced accordingto the invention and other components the amount and composition of which depend on the purpose and technical or scientific field within which the enzyme compositions are to be used. When the enzyme preparations or compositions according to the invention are in solid form they can consist of granules into which the enzymes are incorporated, for instance together with other enzymes or substances having other than enzymatic activity useful for the utility of the enzyme compositions. When the enzymes are not used in crystalline form, they may be accompanied by impurities of organic nature, such as proteins and carbohydrates from the culture medium.

The enzyme compositions in liquid form can constitute solutions or suspensions which may contain stabilizers, if necessary.

Usually, the novel enzymes of the invention are used in small quantities. In view thereof the enzyme preparations or compositions for industrial use normally show an enzyme content not exceeding about 10 percent by weight.

The novel enzymes according to the invention can for instance be used in washing compositions, dehairing compositions, in preparations for hydrolysis of proteins, in dish-washing compositions and as additives to septic tanks and installations for purifying sewage.

Quite a number of tests have been carried out in connection with the utility of the enzyme preparations according to the invention in washing compositions and washing processes.

The detergent which should represent a heavy duty detergent had the following composition: Hereinafter referred to as Detergent Formulation A Nansa S (40% sodiumalkylaryl sulfonate. 60% sodium sulphate) 250 g Nonyl phenol, 10 E0 g Soap (80%) 30 g Sodiumtripolyphosphate 300 g CMC (6O 16 g 30 Sodiumcarbonate, anhydr. 80 g Sodiumsulphate, anhydr. 74 g Sodiumperborate (NaBO ,4 H2) 220 g Total 1000 g The other conditions were as follows:

Hardness of water in German units I0 Fabric to water ratio Time of experiment 30 minutes pl-I-value about 10 Detergent concentration: 4.0 g per liter of washing solution The washing process carried out at 50C was as follows:

By means of a pipette 20 ml of the enzyme solution with an activity of 0.288 Anson units per liter were added to a 150 ml beaker. Beforehand, the pH-value was adjusted to 10.0 and the temperature to 20C. At zero time there were added 100 ml detergent solution adjusted to pH 10.3 and 56C. The concentration of the detergent was 4.8 g per liter. The beaker was immediately placed in a water thermostate at C and 6 circular EM PA-test swatches were added with a total weight of 3.0 g. Agitation was effected with a glass spatula during 10 seconds and the beaker was standing in the thermostate 30 minutes in total, agitation being effected during 10 seconds every 4 minutes. Then the wash solution was separated, and the pH-value was measured after cooling. The test swatches were rinsed in running tap water during 10 minutes and then dried between two towels and ironed. Every test swatch was subjected to remission measurements in a Beckman spectrophotometer at 460 mu.

In addition to these experiments control experiments were carried out in which the enzyme solution was replaced by water.

In the experiments carried out at 60C the temperature of the detergent solution was adjusted to 68C.

The results of these experiments are compiled in the following table X Nonylphenol, 10 E 80 g Sodiumtripolyphosphate 400 g CMC (45%) 20 g Sodium carbonate, anhdrf 150 g Sodium sulphate. anhydr. 350 g Total 1000 g Further conditions:

Water German hardness Fabric to water ratio 1:40 Period 30 minutes Temperature 45 C pH about 10 Detergent concentration 4.0 g per liter of wash solution The procedure was as reported in the washing experiments mentioned above, except that the wash solution had a temperature of 50C before it was mixed with the enzyme solution.

There were carried out three experiments with each of the enzymes, and the results appear from the following Table XI. I

TABLE X1 Enzyme Remission of EMPA-test swatch No. 116 from strain untreated after soaking and rinsing None 16.4 18.7 C 303 36.8 C 339 32.3 C 351 31.0 307 34.3. C 377 37.6 C 364 34.6 C 372 43.2 C 376 36.8 C 366 37.3

The remission values are average figures of the measurements of each of the six test swatches in each of the experiments, except that the values in connection with the experiments with enzymes from strain C 339 and C 351 are based on one measurement.

Experiments have also been carried out while using different enzyme concentrations in the wash solution, viz. in the interval between 0.02 and 0.16 Anson units per liter of wash solution. For the experiments EMPA test swatches No. 1 16 were used and the detergent was the same as that used in connection with the first men-- tioned washing experiments, viz.

Nansa S (40% sodiumalkylarylsulphonate,

60% sodiumsulphatc 250 g Nonylphenol, 10 E0 30 g Soap 30 g Sodiumtripolyphosphate 300 g CMC (60%) 16 g Sodiumcarbonate, anhydr. 80 g Sodiumsulphate. anhydr. 74 g Sodiumperborate (NaBO A H 0 220 g Total 1000 g The test conditions were as follows:

Water 10 German hardness Fabric to water ratio 1:40

Period 30 minutes Temperature 45C and 60C pH about 10 Detergent concentration 4.0 g per liter of wash solution The procedure is the same as that reported in the first mentioned experiments, except that the concentration of the enzyme solution was varied so that the activity in the wash solution gets the following values:

0.02 0.04 0.08 0.16 Anson units per liter Before the detergent solution was mixed with the enzyme solution it was adjusted to 50C and 68C, respectively, corresponding to a temperature of 45C and 60C, respectively, in the wash solution ready for use.

Each enzyme preparation was tested at both temperatures. The results of the remission measurements are collected in the following Table X11.

TABLE X11 Remission of EMPA-test swatches l 16 Enzyme Temperature Anson units per liter of wash solution from strain C 0 0.02 0.04 0.08 0.16 C 303 45 15.0 25.8 28.2 33.5 36.8 C 339 45 16.02 25.8 29.7 32.5 36.0 C 351 45 15.7 26.0 30.8 34.2 37.3 C 367 45 14.0 25.2 28.3 31.7 36.3 C 377 45 16.5 29.2 32.2 36.8 41.7 C 364 45 15.3 27.2 29.3 33.7 36.7 C 372 45 16.2 33.0 36.6 42.0 46.1 C 376 45 17.5 30.0 33.7 36.3 39.8 C 366 45 16.7 31.8 34.0 38.3 46.7 C 303 60 19.2 29.8 32.0 34.1 36.1 C 339 60 16.9 20.9 21.7 25.4 28.8 C 351 60 17.4 27.0 28.8 32.4 34.4 C 367 60 17.3 28.8 30.3 32.6 34.1 C 377 60 17.4 27.0 28.8 32.4 34.4 C 364 60 20.8 28.7 29.8 33.0 33.5 C 372 60 17.0 21.3 23.7 25.7 29.1 C 376 60 19.0 28.4 31.4 33.6 35.5 C 366 60 19.0 32.0 32.5 35.0 38.9

The remission values are average figures from the six measurements. Finally, there have been carried out the following Storage stability experiments (shelflife) Each enzyme preparation was mixed in a Turbola The enzyme Preparations or compositions according mixer with Detergent Formulation A. Corresponding to the invention for use in the detergent industry may experiments were carried out with the same detergent, also contain other enzymes showing utility in the Washexcept that the perborate was substituted by anhydrous g P Quite a num r of uch enzymes are sodium sulphate. The water content in the detergent known in the not containing perborate was 2.7 percent. The water when the enzyme Preparations or eornposltlons eontent in the d t nt ont i i rb t was cording to the invention are intended for use as an acnaturally correspondingly higher because the perborate tive constituent of detergent compositions, the enzyme contains about 47 percent water of crystallization. preparations are usually marketed as a powder, of

All the mixtures were analyzed for proteolytic activiwhich the active enzyme or enzymes constitute a minor ty immediately after they were made and then placed in amount, the balance of the powder consisting of inorsealed glass containers at 40C. The analysis for ganic salts, such as sodium sulphate, calcium phosphate proteolytic activity was repeated with certain intervals. and sodium chloride, sometimes together with other The analyses were carried out as follows: substances forming constituents ofthe final detergents.

From 12 different places in the glass containers there As proteolytic enzymes have already found use as was taken a sample weighing 12.5 g which was transconstituents of detergent compositions, all persons ferred to a 1 liter volumetric flask. in respect ofthe perskilled in the art will know how to make the enzyme borate-containing mixture there were added 4.4 g sodipreparations or compositions according to the invention suited for use in detergent compositions;

It is an essential advance for use in detergent comum sulphite for the purpose of neutralizing the perborate. Deionized water was added up to the mark, and

the solution was then kept for minutes at 25C while positions that many of the enzymes according to the instirring mechanically. Then the proteolytic activity was vention show optimal proteolytic activity at elevated determined in Anson units. pH-values and improved stability in the presence of The mixtures tested and the residual proteolytic ac- 25 perborates. tivity measured at the various periods of storage have As it has been mentioned in the foregoing the enbeen compiled in the following Table XIII. zyme preparations or compositions according to the in- TABLE XIII.STO RING PE RIODS Anson units per gram of enzyme and percentage of activity after storing at C. iii- 0 days 1 day 3 days 5 days 8 days 14 days 21 days 28 days Detergent plus en- Act, Act. Act, Act, Act, Act, Act, Act, znne from Perboperperperperperpcrpcr- Derstrain rate AU/g. cent AU/g. cent AU/g. cent A U/g. cen AU/g. cent AU/g. cen AU/g. cent AU/g. cent NOTE.A U/g.=Anson units per gram; Act. pcrccnt=Percentugc of residual proteolytic activity.

The stability of some of the enzymes in the presence 5 vention can also be used for the purpose of dehairing of perborate is considerable. skins and hides. in the old dehairing process the hides Many of t enzym according to the invention are were placed in a bath containing calcium hydroxide useful not only in the detergent compositions referred d di l hid d h i H- l f bout to in the above experiments but actually useful in 12. This dehairing process is detrimental to the hairs kinds of detergent compositions. Such compositions hi h i h b ofeommereialvalue may contain water-soluble soaps, anionic synthetic de- 55 D i h recent years use h b d of an tergents, such as water-soluble salts of organic sulphuzymatic d h i i process, in hi h u ha be n made ric reaction products, non-ionic synthetic detergents, f proteolytic enzymes, d h d h i i h b Such 88 comp un Produced y Condensation of ried out at a lower pH-value of 7 to 10, which does not kylene oxide groups with an organle hydrophobic 60 affect the quality of the hairs. On the other hand, no P ampholyrle synthetic detergents and other material swelling of the skins or hides is obtained as it synthetic dete g nts- AlSO builders y be Present in was in the old dehairing process, which makes difficulthe detergents and the enzymes according to th cX- ties in the further processing ofthe skins or hides. periments may be combined with such builders. Exam- These difficulties are known in the art, and it has P 0f builders are cafbonares, boffltes, ph p 65 been proposed to use proteolytic enzymes showing sufpolyphosphates, silicates and sulphates of the alkali ficient activity atahigher pH-value than 10.

metals, preferably sodium. As some of the enzymes according to the invention Also organic alkaline builders may be present and show optimal proteolytic activity at a pH-value up to combined with the enzymes according to the invention, 12, these enzymes are well suited for use in dehairing ifconvenient. processes. The following experiments are intended to illustrate the utility of some of the enzymes according to the invention for dehairing.

A salted cowhide (the butt) is sliced in pieces measuring about X 4 cm. The pieces are steeped 24 hours and fat and meat are scraped off. The pieces of hide are then placed in 400 ml of different enzyme solutions contained in glasses having a volume of 500 7 ml. The glasses are incubated at 30C for 24 hours. The

pieces are then removed from the solutions and the hairs are scraped off with a piece of plexiglass. The dehairing effect is evaluated in accordance with the following scale:

1. Easy and complete removal of the hairs.

2. Easy removal of the hairs, but spots of hairs remained on the hide.

3. No or difficult removal of the hairs.

The proteolytic enzyme solutions used contain 1 g calcium hydroxide per 130 g of water. The amount of enzymes appears from the below Table XIV, also indicating the pH-value's at the beginning and the end of the dehairing process together with the results thereof.

TABLE XIV What we claim is:

1. An enzyme preparation consisting of or containing at least one proteolytic enzyme of the serine type derived from the genus Bacillus and showing optimal proteolytic activity at a pH value above about 9 and retaining to percent of maximum proteolytic activity at pH 12, said activities being measured against hemoglobin by the Anson method.

2. An enzyme preparation according to claim 1 wherein the proteolytic enzymes is derived from a bacterium selected from the group consisting of Bacillus species NCIB Nos. 10144 through 10148, 10281, 10284, 10286, 10288, 10301, 10304, 10306 through 10313 and 10315 through 10327.

3. A process for producing a proteolytic enzyme of the serine type showing optimal proteolytic activity against hemoglobin at a pH value above 9 and showing 80 to 100 percent of maximum proteolytic activity at pH 12 when said activities are measured by the Anson method the said method comprising aerobic cultivation of a member of the genus Bacillus selected from the group NCIB Nos. 10144 through 10148,'10281, 10284, 10286, 10288, 10301, 10304, 10306 through 30313 and 10315 through 10327 in a nutrient medium for production of proteolytic enzymes comprising assimilable carbon and nitrogen sources and maintaining the pH value of said nutrient medium between 7.5 and 10.5 during the main part of cultivation.

4. The method of hydrolyzing protein material which comprises contacting the protein material with an aqueous medium containing an enzyme preparation according to claim 1.

rOo

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PATENT NO. 3,723,250 DATED March 27, 1973 INVENTOR(S) Knud Aunstrup et al It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

In the drawings, Figures 1, 2 and 3, "Aktivity" should read Activity- Table 3, the column caption reading "Growth on nitrate (NO as sole N-source" should read Growth on nitrate (N0 as sole N-source:-

Column 21, line 11, amend "plexiglass." to read Plexiglas (a registered trademark of Rohm & Haas Company).

Signed and Scaled this Thirteenth Day Of February 1979 [SEAL] A nest:

RUTH C. MASON D ALD W. BANNER Arresting Officer Commissioner of Patents and Trademarks Disclaimer 3,723,250."Kmui Aunstmp, Farum, Otto Andv'esen, Copenhagen, and Belle Outtmp, Vaerlose, Denmark. PROTEOLYTIC ENZYMES, THEIR PRODUCTION AND USE. Patent dated Mar. 27, 197 3. Disclaimer filed May 1, 1974, by the assignee, Nova Tempeutz'sk Labm'atom'um A/S.

Hereby disclaims the portion ofthe term of the patent subsequent to Jul 4, 1989.

[Ofiicz'al Gazette November 26, 1.974.] 

2. An enzyme preparation according to claim 1 wherein the proteolytic enzymes is derived from a bacterium selected from the group consisting of Bacillus species NCIB Nos. 10144 through 10148, 10281, 10284, 10286, 10288, 10301, 10304, 10306 through 10313 and 10315 through
 10327. 3. A process for producing a proteolytic enzyme of the serine type showing optimal proteolytic activity against hemoglobin at a pH value above 9 and showing 80 to 100 percent of maximum proteolytic activity at pH 12 when said activities are measured by the Anson method the said method comprising aerobic cultivation of a member of the genus Bacillus selected from the group NCIB Nos. 10144 through 10148, 10281, 10284, 10286, 10288, 10301, 10304, 10306 through 30313 and 10315 through 10327 in a nutrient medium for production of proteolytic enzymes comprising assimilable carbon and nitrogen sources and maintaining the pH value of said nutrient medium between 7.5 and 10.5 during the main part of cultivation.
 4. The method of hydrolyzing protein material which comprises contacting the protein material with an aqueous medium containing an enzyme preparation according to claim
 1. 